Download The Plant Membrane-Associated REMORIN1.3 Accumulates in

The Plant Membrane-Associated REMORIN1.3
Accumulates in Discrete Perihaustorial Domains and
Enhances Susceptibility to Phytophthora infestans1[W]
Tolga O. Bozkurt, Annis Richardson, Yasin F. Dagdas, Sébastien Mongrand, Sophien Kamoun,
and Sylvain Raffaele*
Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom (T.O.B., A.R., Y.F.D., S.K., S.R.); Department of
Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom (T.O.B.); John Innes Centre,
Norwich NR4 7UH, United Kingdom (A.R.); Laboratoire de Biogenèse Membranaire, Unité Mixte de Recherche
5200 Centre National de la Recherche Scientifique-Université Bordeaux Segalen-Institut National de la
Recherche Agronomique, F–33883 Villenave d’Ornon cedex, France (S.M.); and Laboratoire des Interactions
Plantes-Microorganismes, Unité Mixte de Recherche 441 Institut National de la Recherche Agronomique-Unité
Mixte de Recherche 2594 Centre National de la Recherche Scientifique, F–31326 Castanet-Tolosan, France (S.R.)
Filamentous pathogens such as the oomycete Phytophthora infestans infect plants by developing specialized structures termed
haustoria inside the host cells. Haustoria are thought to enable the secretion of effector proteins into the plant cells. Haustorium
biogenesis, therefore, is critical for pathogen accommodation in the host tissue. Haustoria are enveloped by a specialized hostderived membrane, the extrahaustorial membrane (EHM), which is distinct from the plant plasma membrane. The mechanisms
underlying the biogenesis of the EHM are unknown. Remarkably, several plasma membrane-localized proteins are excluded
from the EHM, but the remorin REM1.3 accumulates around P. infestans haustoria. Here, we used overexpression, colocalization
with reporter proteins, and superresolution microscopy in cells infected by P. infestans to reveal discrete EHM domains labeled
by REM1.3 and the P. infestans effector AVRblb2. Moreover, SYNAPTOTAGMIN1, another previously identified perihaustorial
protein, localized to subdomains that are mainly not labeled by REM1.3 and AVRblb2. Functional characterization of REM1.3
revealed that it is a susceptibility factor that promotes infection by P. infestans. This activity, and REM1.3 recruitment to the
EHM, require the REM1.3 membrane-binding domain. Our results implicate REM1.3 membrane microdomains in plant susceptibility
to an oomycete pathogen.
Filamentous plant pathogens, including oomycetes
of the genus Phytophthora, downy mildews and white
rusts, as well as powdery mildews and rust fungi, are
among the most devastating plant pathogens. These
biotrophic parasites associate closely with plant cells
through specialized infection structures called haustoria. Haustoria are specialized pathogen hyphal
structures formed within host cells and enveloped by
a perimicrobial membrane called the extrahaustorial
membrane (EHM), a key interface between plant pathogens and the host cell. Haustoria are critical for
successful parasitic infection by many filamentous
plant pathogens and are a signature of the biotrophic
1
This work was supported by the Gatsby Charitable Foundation,
the European Research Council, the Biotechnology and Biological Sciences Research Council, and by Marie Curie Intra European Fellowships (contract no. 268419 to T.O.B. and contract no. 255104 to S.R.).
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Sylvain Raffaele ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.114.235804
lifestyle. In fungi, haustoria function as feeding structures
(Voegele et al., 2001). In addition, haustoria are thought to
enable the delivery of host-translocated virulence proteins, known as effectors, by both fungal and oomycete
pathogens (Catanzariti et al., 2006; Whisson et al., 2007).
However, little is known about the molecular mechanisms underlying the biogenesis and function of haustoria and EHM (Kemen and Jones, 2012; Lu et al., 2012).
The EHM is thought to be continuous with the host
plasma membrane (PM), yet it is a highly specialized
membrane compartment that develops only in plant
cells that accommodate haustoria (haustoriated cells;
Coffey and Wilson, 1983). On the plant side, all eight
PM proteins tested by Koh et al. (2005) were excluded
from the EHM in Arabidopsis (Arabidopsis thaliana)
cells infected with the powdery mildew fungus Golovinomyces cichoracearum. Conversely, the atypical Arabidopsis resistance protein Resistance to Powdery
Mildew8.2 (RPW8.2) exclusively localizes to the EHM
in this interaction (Wang et al., 2009). Ultrastructure
analyses of the Golovinomyces orontii powdery mildew
pathosystem revealed that the EHM is asymmetric,
thicker and more electron opaque than the PM, and
can be highly convoluted around mature haustoria (Micali
et al., 2011). More recently, a survey of Arabidopsis and
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1005
Bozkurt et al.
Nicotiana benthamiana plants infected by the oomycete
pathogens Hyaloperonospora arabidopsidis and Phytophthora
infestans, respectively, revealed that several integral
host PM proteins are excluded from the EHM (Lu et al.,
2012). Nevertheless, the remorin REM1.3 and the
SYNAPTOTAGMIN1 (SYT1) peripheral membrane proteins localized to undetermined subcellular compartments around haustoria in P. infestans-plant interactions
(Lu et al., 2012). Whether the differential accumulation
of membrane proteins at the EHM is due to interference with the lateral diffusion of proteins from the PM
or targeted secretion of specialized vesicles remains unclear (Lu et al., 2012).
The subcellular distribution of effectors inside plant
cells provides valuable clues about the host cell compartments they modify to promote disease, and effectors have emerged as useful molecular probes for plant
cell biology (Whisson et al., 2007; Bozkurt et al., 2012).
Heterologous expression of fluorescently tagged effectors in plant cells has been used to determine their
subcellular localization in uninfected and infected tissue. This approach has been successful with the RXLR
and CRINKLER (CRN) effectors, the two major classes
of cytoplasmic (host-translocated) oomycete effectors
(Bozkurt et al., 2012). The 49 H. arabidopsidis RXLR effectors studied by Caillaud et al. (2012) localized to the
nucleus, the cytoplasm, or various plant membrane
compartments. In contrast, CRN effectors from several
oomycete species exclusively accumulate in the plant
cell nucleus (Schornack et al., 2010; Stam et al., 2013).
The P. infestans effectors AVRblb2 and AVR2 accumulate around haustoria when expressed in infected
N. benthamiana cells, highlighting the PM and the EHM
as important sites for effector activity (Bozkurt et al.,
2011; Saunders et al., 2012). These effectors, therefore,
can serve as useful probes for plant cell biology to
dissect vesicular trafficking and focal immunity, processes that have proved difficult to study using standard
genetic approaches (Bozkurt et al., 2011; Win et al.,
2012).
REM1.3 is one of two plant membrane-associated
proteins detected around haustoria during the interaction between P. infestans and the model plant
N. benthamiana (Lu et al., 2012). Therefore, we hypothesized that studying REM1.3 should prove useful for
understanding the mechanisms governing the function
and formation of perihaustorial membranes. REM1.3
belongs to a diverse family of plant-specific proteins containing a Remorin_C domain (PF03763) and
has known orthologs in potato (Solanum tuberosum;
StREM1.3), tomato (Solanum lycopersicum; SlREM1.2),
tobacco (Nicotiana tabacum; NtREM1.2), and Arabidopsis (AtREM1.1–AtREM1.4; Raffaele et al., 2007).
Several proteins from the remorin family, including
REM1.3, are preferentially associated with membrane
rafts, nanometric sterol- and sphingolipid-rich domains in PMs (Pike, 2006; Simons and Gerl, 2010).
Indeed, StREM1.3 and NtREM1.2 are highly enriched
in detergent-insoluble membranes (DIMs) and form
sterol-dependent domains of approximately 75 nm in
1006
purified PMs (Mongrand et al., 2004; Shahollari et al.,
2004; Raffaele et al., 2009). StREM1.3 directly binds to
the cytoplasmic leaflet of the PM through a C-terminal
anchor domain (RemCA) that folds into a hairpin of
aliphatic a-helices in polar environments (Raffaele et al.,
2009; Perraki et al., 2012). StREM1.3 is differentially
phosphorylated upon the perception of polygalacturonic
acid (Reymond et al., 1996). AtREM1.3 is differentially
recruited to DIMs and differentially phosphorylated
upon flg22 (for flagellin) peptide perception (Benschop
et al., 2007; Keinath et al., 2010; Marín et al., 2012), suggesting a role in plant defense signaling. StREM1.3 and
SlREM1.2 prevent Potato virus X spreading by interacting
with the Triple Gene Block protein1 (TGBp1) viral movement protein, presumably in plasmodesmata or at the PM
(Raffaele et al., 2009; Perraki et al., 2012). AtREM1.2 belongs to protein complexes formed by a negative regulator
of immune responses, Resistance to Pseudomonas syringae
pv maculicola1 (RPM1)-INTERACTING PROTEIN4, at the
PM (Liu et al., 2009). Furthermore, Medicago truncatula
MtSYMREM1 is enriched in root cell DIMs (Lefebvre
et al., 2007) and localizes to patches at the peribacteroid
membrane during symbiosis with Sinorhizobium meliloti
(Lefebvre et al., 2010). MtSYMREM1 is important for
nodule formation and interacts with the Lysin motif
domain–containing receptor-like kinase3 (LYK3) symbiotic receptor (Lefebvre et al., 2010). Multiple lines of
evidence, therefore, implicate several remorins in cell
surface signaling and the accommodation of microbes
during plant-microbe interactions (Raffaele et al., 2007;
Jarsch and Ott, 2011; Urbanus and Ott, 2012). Nevertheless, little is known about REM1.3’s molecular function,
and its role in immunity against filamentous plant pathogens has not been reported to date.
In this study, we analyzed in detail the localization
and function of REM1.3 during host colonization by
P. infestans. We found that REM1.3 localizes exclusively
to the vicinity of the PM and the EHM around noncallosic haustoria. Furthermore, our results suggest that
the EHM is likely formed by multiple microdomains.
REM1.3 silencing and overexpression experiments demonstrated that it promotes susceptibility to P. infestans
in N. benthamiana and tomato. We also show that the
REM1.3 membrane anchor domain is required for its
localization at the EHM and for the promotion of susceptibility to P. infestans. This work demonstrates the
importance of the dynamic reorganization of the PM in
response to haustoria-forming pathogens. Our study also
revealed that the effector AVRblb2 localizes to remorincontaining host membrane domains at the host-pathogen
interface, possibly as a pathogen strategy to facilitate the
accommodation of infection structures inside plant cells.
RESULTS
REM1.3 Localizes at the PM and the EHM in Cells Infected
by P. infestans
N. benthamiana is a versatile host system in which to
study the cellular and molecular dynamics of the plant
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REMORIN1.3 Perihaustorial Domains and Disease Susceptibility
response to the hemibiotrophic pathogen P. infestans
(Chaparro-Garcia et al., 2011; Lu et al., 2012). Using
fluorescent markers for the cytoplasm, tonoplast, and
EHM, it was found that these three subcellular compartments occur closely around P. infestans haustoria
(Bozkurt et al., 2012; Caillaud et al., 2012), which
makes the distinction between the EHM and these
other compartments challenging. To determine in
which perihaustorial compartment REM1.3 resides,
we performed a series of colocalization studies using various marker proteins labeling distinct perihaustorial compartments in N. benthamiana plants
inoculated by P. infestans. First, to determine the extent
to which REM1.3 localizes to the EHM or the cytoplasm surrounding the EHM, we coexpressed red
fluorescent protein (RFP):REM1.3 and GFP in infected
plant cells by Agrobacterium tumefaciens-mediated
transient transformation under the control of the
cauliflower mosaic virus (CaMV) 35S promoter. At
4 d post inoculation (dpi), RFP:REM1.3 surrounded
P. infestans haustoria tightly and showed a sharp and
focused signal in contrast to the diffuse cytosolic
localization pattern of free GFP, suggesting that
REM1.3 localizes specifically at the EHM (Fig. 1A).
Second, to exclude the possibility that REM1.3 accumulates at the tonoplast surrounding the EHM,
we coexpressed RFP:REM1.3 with the H. arabidopsidis
effector HaRXL17, which marks the perihaustorial
tonoplast in host cells challenged with P. infestans
(Bozkurt et al., 2012; Caillaud et al., 2012; Fig. 1B). At 4
dpi, RFP:REM1.3 fluorescence was tightly surrounded
by GFP:HaRXL17 fluorescence. In plots measuring
fluorescence along a line cutting through a haustorium, the two peaks of RFP:REM1.3 fluorescence
were located between the two peaks of GFP:HaRXL17,
indicating that REM1.3 localizes between the tonoplast and the haustorium. Finally, we coexpressed
yellow fluorescent protein (YFP):REM1.3 with the
P. infestans RXLR effector AVRblb2 that associates with
the EHM in infected plant cells (Bozkurt et al., 2011,
2012; Fig. 1C). Remarkably, REM1.3 and AVRblb2
colocalized almost completely around the haustorium, further highlighting the association of REM1.3
with the EHM.
Figure 1. REM1.3 localizes at the
PM and the EHM in cells infected
by P. infestans. Coexpression of
RFP:REM1.3 and GFP (A), RFP:
REM1.3 and GFP:HaRXL17 (B),
and YFP:REM1.3 and RFP:AVRblb2
(C) by A. tumefaciens-mediated
transient transformation under the
control of the CaMV 35S promoter
in haustoriated cells discriminates
between host subcellular compartments surrounding haustoria.
GFP, HaRXL17, and AVRblb2 label the cytoplasm and nucleus,
the tonoplast, and the EHM and
PM, respectively. Colocalization is
only observed between REM1.3
and AVRblb2. Images show single
optical sections. The fluorescence
plots show relative fluorescence
along the dotted line connecting
points a and b. Arrowheads point
to the tips of haustoria. A.U., Arbitrary units; Cyt., cytoplasm; Ton.,
tonoplast. Bars = 7.5 mm.
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Bozkurt et al.
REM1.3 Accumulates around Noncallosic Haustoria during
Infection by P. infestans
To document the robustness and dynamics of
REM1.3 localization at the EHM, we used N. benthamiana transgenic plants constitutively expressing
YFP:REM1.3 (Lu et al., 2012). We inoculated these
plants with the transgenic P. infestans isolate 88069
constitutively expressing RFP (88069td; ChaparroGarcia et al., 2011) and monitored the distribution of
YFP:REM1.3 in infected cells. We observed in approximately 50% (102 out of 197 cases) that haustoria
were surrounded by REM1.3, while in the rest of the
cases, REM1.3 only remained at the adjacent PM
(Fig. 2A). To further analyze REM1.3 localization at the
EHM, we compared its perihaustorial distribution
with that of AVRblb2. When coexpressed, YFP:
REM1.3 was detected at the EHM in nearly 50% of the
cases (seven out of 16 observations), while RFPAVRblb2 always localized at the EHM (16 out of 16;
Fig. 2B).
Unlike the haustoria of powdery mildew fungi and
H. arabidopsidis, P. infestans haustoria are rarely surrounded by callose encasements but sometimes accumulate a callosic collar (Bozkurt et al., 2011). Callose
encasements are thought to be indicative of a plant
defense reaction and do not reflect an active and
functional haustorial interface (van Damme et al.,
2009). To determine the degree to which REM1.3 perihaustorial accumulation is associated with callose, we
performed aniline blue staining on plants expressing
YFP:REM1.3 and infected by a P. infestans strain
(88069td) expressing a cytosolic RFP. REM1.3-labeled
haustoria did not display a callosic collar (Fig. 2C),
indicating that the perihaustorial localization of
REM1.3 is not due to encasement of the haustoria. This
suggests that REM1.3 could play a role in the control of
the infection process via the suppression of callose
deposition or other unknown mechanisms.
REM1.3 Colocalizes with the P. infestans RXLR Effector
AVRblb2 in Specific Domains at the EHM
REM1.3 is a well-established protein marker of sterol- and sphingolipid-rich PM domains designated as
membrane rafts (Raffaele et al., 2009). We observed
that REM1.3 displays nonuniform perihaustorial accumulation, delimiting discrete membrane domains at
the EHM (Fig. 1). Similar to REM1.3, the P. infestans
RXLR effector AVRblb2 localizes to the PM and
dramatically relocalizes to the EHM during host infection (Bozkurt et al., 2011, 2012). We observed that
REM1.3 and AVRblb2 significantly colocalize at the
EHM in haustoria cross sections (Fig. 1). To test
whether AVRblb2 specifically targets REM1.3containing membrane domains, we examined the degree to which these two proteins colocalize in haustoria
longitudinal sections. For this, we coexpressed YFP:
REM1.3 and RFP:AVRblb2 in N. benthamiana-infected
cells at 4 dpi. Both RFP:AVRblb2 and YFP:REM1.3
1008
Figure 2. REM1.3 localizes around a subpopulation of noncallosic
haustoria during P. infestans infection. A, Representative image of a
stable 35S-YFP:REM1.3 N. benthamiana transgenic plant inoculated
by P. infestans strain 88069 expressing RFP (88069td). Haustoria are
indicated with closed arrowheads when surrounded by YFP:REM1.3
and with open arrowheads otherwise. B, Frequency of the colocalization of YFP:REM1.3 with RFP:AVRblb2 around P. infestans haustoria. YFP:REM1.3 with RFP:AVRblb2 constructs were codelivered into
N. benthamiana leaves by A. tumefaciens-mediated transformation. C,
P. infestans 88069td-inoculated leaves expressing YFP:REM1.3 and
stained for callose. Haustoria surrounded by YFP:REM1.3 (closed arrowheads) never showed a callose neck band (open arrowheads).
Images show single optical sections. The frequency of observations is
indicated.
distributed heterogenously around haustoria-forming
perihaustorial foci. The more intense RFP:AVRblb2
foci colocalized with YFP:REM1.3 foci (Fig. 3A, arrowheads). To quantify the degree of REM1.3 and
AVRblb2 colocalization, we extracted fluorescence
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REMORIN1.3 Perihaustorial Domains and Disease Susceptibility
signals along the EHM and calculated Pearson correlation coefficients (r values; Fig. 3B). The average
Pearson correlation coefficient between the YFP and
RFP fluorescence signals around haustoria was 0.79,
indicating that REM1.3 and AVRblb2 are present at the
same perihaustorial domains.
To further define the membrane perihaustorial domains, we coexpressed REM1.3 and AVRblb2 with
Figure 3. REM1.3 colocalizes with the P. infestans RXLR effector AVRblb2 in domains around haustoria. A, Coexpression of EHM
markers by A. tumefaciens-mediated transient transformation under the control of the CaMV 35S promoter in haustoriated cells reveals
perihaustorial membrane domains. Left, YFP:REM1.3 and RFP:AVRblb2 show nearly full colocalization around haustoria, with all
domains strongly labeled by YFP:REM1.3 (closed arrowheads) showing intense RFP fluorescence. Middle, SYT1 strongly labels domains that are only weakly labeled by RFP:AVRblb2 (open arrowheads). Right, SYT1 labels domains around haustoria that are not or
weakly labeled by YFP:REM1.3 (open arrowheads). B, Correlation between the RFP and YFP fluorescence signals around haustoria in
cells coexpressing RFP:AVRblb2 and YFP:REM1.3. The fluorescence is measured along the dotted line connecting points a and b, as
shown in the inset. The average Pearson correlation coefficient for RFP and YFP fluorescence signals along six different perihaustorial
membranes is 0.79. A.U., Arbitrary units. C, Quantification of fluorescence correlation for perihaustorial markers highlights the existence of at least two types of domains around haustoria. Pearson correlation coefficients were calculated in cells coexpressing free
GFP+RFP:REM1.3, YFP:REM1.3+RFP:AVRblb2, GFP:SYT1+RFP:AVRblb2, and GFP:SYT1+RFP:REM1.3. Only cells in which REM1.3
accumulated around haustoria were considered. Significant differences of the means were assessed using Welch’s t test (***P ,
0.001). Measurements were performed over at least two independent inoculation events.
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Bozkurt et al.
SYT1, another plant PM-associated protein localized
around haustoria (Lu et al., 2012). SYT1 localized in
foci around haustoria mostly distinct from foci labeled
by AVRblb2 and REM1.3 (Fig. 3A). Since our aim was
to characterize perihaustorial domains, we focused the
next steps of the analysis on haustoriated cells in
which REM1.3 accumulated around haustoria. First,
to estimate the background correlation associated
with the bleed through of fluorescence, we calculated r
for the fluorescence signals along haustoria in cells
expressing free GFP and RFP:REM1.3. We obtained an
average r of approximately 0.1, indicating that the
GFP and REM1.3 do not colocalize along haustoria
(Fig. 3C). Second, we calculated r for cells expressing
YFP:REM1.3 and RFP:AVRblb2 and obtained an average r of approximately 0.8. Third, we calculated r for
cells expressing GFP:SYT1 and RFP:AVRblb2 as well
as RFP:SYT1+YFP:REM1.3. We obtained average r
values of approximately 0.4 and 0.5, respectively. To
test whether colocalization between YFP:REM1.3 and
AVRblb2:RFP was significantly higher than between free
GFP and RFP:REM1.3, GFP:SYT1 and RFP:AVRblb2, or
RFP:SYT1 and YFP:REM1.3, we used Welch’s t test. We
obtained P , 0.001, indicating that correlation between
YFP:REM1.3 and AVRblb2:RFP can be considered higher
than the others with 99.9% confidence.
Superresolution Structured Illumination Microscopy
Confirms the Occurrence of REM1.3 Microdomains
at the EHM
To discriminate subcellular compartments accumulating around haustoria and validate the observation
of different domains at the EHM with a resolution of
approximately 100 nm, we conducted similar experiments using superresolution imaging by structured
illumination microscopy (SIM) in N. benthamiana
(Gutierrez et al., 2010). These experiments again
revealed EHM subdomains colabeled by REM1.3 and
AVRblb2 but differentially labeled by SYT1 (Fig. 4;
Supplemental Movies S1–S3). Altogether, these results
demonstrate the high-resolution colocalization of
REM1.3 and AVRblb2, supporting their localization to
the EHM and the lateral compartmentalization of the
EHM into multiple domains.
REM1.3 Overexpression Increases Susceptibility to
P. infestans in N. benthamiana
The recruitment of REM1.3 to domains around active haustoria prompted us to test whether REM1.3
plays a role in susceptibility to P. infestans. For this,
we analyzed the phenotype of transgenic plants
Figure 4. Validation of the occurrence
of subdomains at the EHM using superresolution microscopy. YFP:REM1.3 and
RFP:AVRblb2 show perfect localization
at the EHM mainly at some foci (top
row), while GFP:SYT1 labels different
microdomains compared with RFP:
REM1.3 (middle row). Consistently,
RFP:AVRblb2 and GFP:SYT1 also label
different domains across the EHM
(bottom row). Recombinant constructs
were delivered using A. tumefaciensmediated transformation. Images were
obtained at 3 dpi using superresolution
SIM. Images shown are maximal projections of 31, 30, and 27 frames with
0.11, 0.11, and 0.12 mm steps for the top,
middle, and bottom rows, respectively.
1010
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REMORIN1.3 Perihaustorial Domains and Disease Susceptibility
constitutively expressing YFP:REM1.3 (overexpression).
We first verified the expression and integrity of the
YFP:REM1.3 fusion protein in these plants using antiremorin western-blot analysis (Fig. 5A). We next
tested the response of these plants to P. infestans
using zoospore solution droplet inoculation. We
counted the proportion of inoculated sites showing
no symptoms, necrotic lesions, or P. infestans sporulation in control (wild-type) and overexpression plants.
We found that the frequency of P. infestans sporulation
correlated with higher REM1.3 accumulation, whereas
the frequency of inoculated areas with no symptom correlated with reduced REM1.3 accumulation
(Fig. 5B). At 5 dpi, lesions caused by P. infestans growth
almost completely covered overexpression plant leaves,
whereas the lesions extended slightly beyond the
zoospore droplets in wild-type plants at this stage
(Fig. 5C). Using image analysis to quantify the surface
occupied by hyphae of P. infestans 88069td, we found
an approximately 1.5-fold increase in overexpression
plants compared with controls (Fig. 5D). We also used
transient A. tumefaciens-mediated overexpression of
YFP:REM1.3 in N. benthamiana. In half-leaves overexpressing REM1.3, the infected area was on average
twice as large as in half-leaves overexpressing GFP
(Fig. 5, E and F), indicating that REM1.3 overexpression
enhanced susceptibility to P. infestans. Quantification
of the fluorescence due to GFP and YFP expression as
well as anti-GFP western-blot analysis performed on
total protein extracts allowed us to select for leaves in
which the two A. tumefaciens-delivered constructs were
expressed to similar levels (Fig. 5G). Taken together,
these results indicate a positive role for REM1.3 in
susceptibility toward P. infestans.
Silencing of REM1.3 Enhances Resistance to P. infestans in
N. benthamiana
Figure 5. REM1.3 overexpression increases susceptibility to P. infestans in N. benthamiana. A, Validation of YFP:REM1.3 overexpression
in N. benthamiana transgenic plants by anti-remorin western blot in
two independent 35S-YFP:REM1.3 lines (OX1.4 and OX2.2) compared
with wild-type plants (WT). B, Type and frequency of symptoms
caused by P. infestans 88069 at 5 dpi on overexpression and wild-type
plants as a percentage of 40 infection foci over three independent
experiments including three independent overexpression lines. C,
Representative images of symptoms caused by P. infestans 88069 on
N. benthamiana overexpression and wild-type plants at 5 dpi. D,
Quantification of P. infestans 88069td growth in N. benthamiana lines
by measurement of RFP fluorescence. Representative fluorescence
images show P. infestans 88069td growth in overexpression and wildtype plants at 4 dpi. Bars = 5 mm. Histograms show relative fluorescence
intensity, calculated as the mean pixel intensity over a 0.655-cm2
image centered on the lesion and expressed as a percentage of the
To further validate the role of REM1.3 in response to
P. infestans, we first conducted a phylogenetic analysis
to identify REM1.3 orthologs in N. benthamiana. We
found three REM1.3 orthologs in the N. benthamiana
genome (Supplemental Fig. S1; Supplemental Data S1).
Then, we used a virus-induced gene silencing (VIGS)
approach to silence the REM1.3 orthologs in N. benthamiana using the tobacco rattle virus pTV00 vector
(Ratcliff et al., 2001; Supplemental Fig. S1). Eighteen
intensity measured on wild-type plants. Three to six images were
analyzed per N. benthamiana line, and error bars show SD. E, N.
benthamiana leaf infiltrated with A. tumefaciens carrying either 35S-GFP
or 35S-YFP:REM1.3 (left and right, respectively) and inoculated with P.
infestans 88069 24 h later. Images were taken and the size of lesions
measured at 5 dpi. F, Relative P. infestans lesion size on half-leaves
infiltrated with 35S-GFP and 35S-YFP:REM1.3. Significance was
assayed using Student’s t test (***P , 0.01) over 12 lesions in three
independent experiments. G, Total proteins extracted from half-leaves
infiltrated with 35S-GFP and 35S-YFP:REM1.3 and probed by anti-GFP
western blots showing similar expression levels for GFP and YFP:
REM1.3 constructs.
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Bozkurt et al.
days after delivery of the remorin-silencing construct,
but not with the empty vector control (pTV00), we
observed a strong decrease in YFP fluorescence in
N. benthamiana plants stably expressing YFP:REM1.3,
validating the efficiency of silencing (Supplemental
Fig. S2). In addition, anti-remorin western-blot analysis of total protein extracts from wild-type and silenced
(VIGS) N. benthamiana plants confirmed the suppression of REM1.3 accumulation by our silencing construct (Fig. 6A). Six-week-old silenced plants did
not show any apparent developmental phenotype
(Supplemental Fig. S2). We then tested the response of
silenced plants to P. infestans using zoospore solution
droplet inoculation. At 5 dpi, approximately 20% of
infection foci in virus-free (wild-type) plants and control plants expressing the pTV00 empty vector showed
sporulation, whereas this proportion was less than 5%
for foci in silenced plants (Fig. 6B). Conversely, although 20% of infection foci in silenced plants did not
show any symptoms, this proportion was reduced to
less than 10% in virus-free and empty vector control
plants. At 7 dpi, confluent lesions caused by P. infestans
growth were clearly visible on control plants, whereas the lesions hardly extended beyond the zoospore droplets in silenced plants (Fig. 6C). To confirm
that lesion size correlates with pathogen growth in
these plants, we used image analysis to quantify the
surface occupied by hyphae of P. infestans 88069td. We
measured an approximately 10-fold decrease in the
surface colonized by P. infestans 88069td in silenced
plants compared with control plants (Fig. 6D).
REM1.3 Promotes Susceptibility to P. infestans in Tomato
Figure 6. Silencing of REM1.3 enhances resistance to P. infestans in N.
benthamiana. A, Validation of the silencing of REM1.3 orthologs in N.
benthamiana by anti-REM western blot. The REM1.3 protein amount
was estimated based on the western-blot signal. B, Type and frequency
of symptoms caused by P. infestans 88069 at 6 dpi as a percentage of
at least 12 infection foci over three independent experiments. C,
Representative images of symptoms caused by P. infestans 88069 on
N. benthamiana at 7 dpi. D, Top, representative fluorescence images
showing P. infestans 88069td growth at 4 dpi. Bars = 5 mm. Bottom,
quantification of P. infestans 88069td growth in N. benthamiana lines
1012
Most cultivated plants in the Solanaceae family, including tomato and potato, are susceptible to P. infestans. To test whether the function of remorin in the
N. benthamiana response to P. infestans is conserved
in economically important crops, we inoculated zoospores of P. infestans on tomato transgenic plants
expressing sense and antisense constructs for the
REM1.3 tomato ortholog (Raffaele et al., 2009). The
level of REM1.3 in individual plants relative to the
wild type was evaluated by anti-remorin western-blot
analysis prior to infection (Supplemental Fig. S3). In
plants overexpressing REM1.3, P. infestans-induced
lesions appeared significantly larger than in wild-type
and control plants (150% of the wild type on average
and up to 300%; Fig. 7). Conversely, plants expressing
an antisense REM1.3 construct showed reduced lesions
(75% of the wild type on average). Statistics calculated
on approximately 50 infection foci per line supported
the conclusion that REM1.3 promotes susceptibility to
P. infestans in tomato. We observed a similar degree of
by measurement of RFP fluorescence. Histograms show relative fluorescence intensity calculated as for Figure 5. Three to six images were
analyzed per N. benthamiana line, and error bars show SD. e.v., Plants
infiltrated with the pTV00 empty vector; VIGS, plants infiltrated with
the REM1.3 VIGS silencing construct; WT, wild-type plants.
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REMORIN1.3 Perihaustorial Domains and Disease Susceptibility
transformed leaves with P. infestans 88069td and observed haustoria formed in transformed cells at 4 and
5 dpi. As reported earlier, a strong YFP accumulation
is visible around approximately 50% of haustoria
formed in YFP:REM1.3-expressing cells. By contrast, a
uniform cytoplasmic YFP localization is seen in YFP:
REM1.3ΔCA- and YFP:REM1.3*-expressing cells, and
none of the haustoria observed in these cells showed
any accumulation of YFP fluorescence (more than 30
haustoria surveyed for each construct; Fig. 8B). Therefore, the RemCA membrane anchor is required for
REM1.3 relocalization around P. infestans haustoria.
The REM1.3 Membrane Anchor Is Required for the
Promotion of Susceptibility to P. infestans
To test whether the REM1.3 membrane-binding
domain is required for the promotion of susceptibility to P. infestans, we measured P. infestans lesion size
Figure 7. The REM1.3 ortholog promotes susceptibility to P. infestans
in tomato. A, Symptoms caused by P. infestans 88069 at 4 dpi on
tomato plants overexpressing a tomato REM1.3 ortholog (SE), empty
vector-transformed plants (e.v.), and wild-type (WT) and REM1.3 antisense (AS) plants. B, Box plot showing the distribution of the relative
sizes of lesions at 4 dpi on tomato plants with different levels of
REM1.3. At least 48 infection foci were measured per line over three
independent experiments. The significance of differences compared
with the wild type was assessed by Student’s t test (***P , 0.01).
Overexpression and silencing of REM were verified by western-blot
analysis of individual plants (Supplemental Fig. S3).
increase in P. infestans infection in N. benthamiana
plants overexpressing YFP:REM1.3 and in tomato
plants overexpressing untagged REM1.3, indicating
that these REM1.3 orthologs have similar functions in
response to P. infestans, the YFP tag does not significantly alter this function, and the molecular mechanisms underlying this function are conserved in
N. benthamiana and tomato.
The REM1.3 Membrane Anchor Is Required for
Relocalization at the EHM
We recently demonstrated that REM1.3 is targeted
to the PM through direct lipid binding of a C-terminal
a-helical domain named RemCA (Perraki et al., 2012).
To test whether REM1.3 PM binding is also required
for relocalization around haustoria, we expressed YFPtagged wild-type and mutant REM1.3 constructs in
N. benthamiana using A. tumefaciens-mediated transformation. Consistent with previous reports, YFP:REM1.3
localized exclusively at the PM, whereas mutants
lacking the RemCA domain (YFP:REM1.3ΔCA) or
mutated in the RemCA domain (YFP:REM1.3*) localized to the cytoplasm in noninfected N. benthamiana
epidermal cells (Fig. 8A). We subsequently inoculated
Figure 8. The REM1.3 membrane-binding domain is required for
perihaustorial targeting. Confocal micrographs show the subcellular
localization of YFP fusions with wild-type REM1.3, REM1.3 lacking the
C-terminal membrane anchor domain (DCA), and REM1.3 with mutated C-terminal membrane anchor domain (*) in uninfected cells (A)
and cells infected by P. infestans 88069td (B). The tips of haustoria are
shown by closed arrowheads when surrounded by YFP labeling and
with open arrowheads otherwise. Constructs controlled by the 35S
promoter were delivered using A. tumefaciens-mediated transformation. Images shown are single optical plane sections except for uninfected cells expressing YFP:REM1.3DCA and YFP:REM1.3*, which
correspond to maximal projections of 32 frames with 1 mm steps.
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Bozkurt et al.
formed on N. benthamiana leaves expressing full-length
or mutated REM1.3 constructs. Half-leaves expressing
YFP:REM1.3 showed lesions approximately 250% the
size of half-leaves expressing the GFP control, whereas
sectors expressing YFP:REM1.3ΔCA or YFP:REM1.3*
showed lesions the same size as on half-leaves infiltrated with the GFP control (Fig. 9). These results indicate that the RemCA membrane anchor is required
for REM1.3 function in susceptibility to P. infestans.
This also suggests that REM1.3 localization to microdomains, either at the PM or at the EHM, is essential
for the promotion of susceptibility to P. infestans.
DISCUSSION
The EHM is a critical interface between eukaryotic
pathogens and plants, yet we know little about its biogenesis, composition, and function. To gain insights
into the biology of the EHM, we investigated the role
of REM1.3, one of two plant membrane proteins
known to accumulate around haustoria during infection of N. benthamiana by P. infestans (Lu et al., 2012).
We used a combination of cell biology and pathology
assays to demonstrate the localization of REM1.3 into
discrete perihaustorial domains that are also labeled
by the P. infestans RXLR effector AVRblb2. Genetic
analyses revealed that REM1.3 enhances P. infestans
colonization and, therefore, can be considered a susceptibility factor (Van Damme et al., 2005; Pavan et al.,
2010). Thus, to our knowledge, REM1.3 is the first
plant susceptibility protein to localize at the haustorial
interface, supporting the view that plant pathogens
are likely to perturb host membrane processes to promote intracellular accommodation inside host cells and
infection.
Although many plant PM proteins are excluded
from the EHM, REM1.3 appears to localize to discrete
domains around haustoria, presumably at the EHM.
Such REM1.3 domains could also reside in the extrahaustorial matrix between the EHM and the oomycete
haustorial cell wall, although this is less likely, since
REM1.3 was shown to localize to the cytoplasmic
leaflet of the plant PM (Raffaele et al., 2009). Indeed,
REM1.3 is a well-established plant membrane raft
marker protein that binds directly to negatively
charged lipids that are enriched in plant membrane
rafts (Raffaele et al., 2009; Furt et al., 2010; Perraki
et al., 2012). The association of REM1.3 with the EHM
suggests that this membrane may have a lipid composition close to that of membrane rafts. Similarly, the
peribacteroid membrane that is formed during bacterial endosymbiosis in plants also shares similarities
Figure 9. The REM1.3 membrane anchor is required for the promotion
of susceptibility to P. infestans. A, Symptoms caused by P. infestans
88069 at 5 dpi on leaves transiently overexpressing YFP fusions with
wild-type REM1.3 on one half and free GFP or REM1.3 lacking the
C-terminal membrane anchor domain (DCA) or REM1.3 with mutated
C-terminal membrane anchor domain (*) on the other half. B, Box plot
showing relative sizes of the lesions over 12 to 54 infection foci in
1014
three independent experiments. Significance of differences compared
with GFP-expressing leaves was assessed by Student’s t test (***P ,
0.01). Constructs controlled by the 35S promoter were delivered using
A. tumefaciens-mediated transformation; their expression was verified
by detection of fluorescence.
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REMORIN1.3 Perihaustorial Domains and Disease Susceptibility
with membrane rafts (Pumplin and Harrison, 2009;
Lefebvre et al., 2010). Bhat et al. (2005) reported that
plant membrane proteins such as the barley (Hordeum
vulgare) MILDEW RESISTANCE PROTEIN O, the
barley syntaxin REQUIRED FOR MLO-SPECIFIED
RESISTANCE2 (ROR2), and the Arabidopsis syntaxin
PENETRATION1 (PEN1) redistribute toward Blumeria
graminis f. sp. hordei penetration sites during infection.
These penetration sites are strongly stained by the filipin
dye, indicating abundance in sterols and leading the
authors to propose that membrane raft-like domains
form below the appressoria of mildew fungi (Bhat et al.,
2005; Bhat and Panstruga, 2005).
Accumulating evidence suggests that pathogens
manipulate host cells to establish perimicrobial domains with a specific lipid composition (Cossart and
Roy, 2010; Ham et al., 2011; Gu and Innes, 2012). What
could be the functional and evolutionary advantages of establishing sterol- and sphingolipid-rich perihaustorial membrane compartments? The altered lipid
composition of the EHM may enable directional
transfer of molecules, nutrients, and effectors through
the pathogen membrane and the host-derived membrane. In addition, sterols and sphingolipids, the major
lipid components of membrane rafts, are very diverse
lipid groups, including several plant-specific forms
(Suzuki and Muranaka, 2007; Pata et al., 2010; Cacas
et al., 2012). These lipids, therefore, may constitute a
signature of the host membrane that haustoria-forming
pathogens evolved to target and manipulate.
The perihaustorial membrane domains containing
REM1.3 colocalize with the P. infestans host-translocated
effector AVRblb2, one of a handful RXLR-type effectors
known to localize around haustoria in infected plant
cells (Bozkurt et al., 2011, 2012; Saunders et al., 2012).
Functional analyses indicated that host membrane
targeting is crucial for the promotion of susceptibility
by AVRblb2 (Bozkurt et al., 2011). AVRblb2 prevents
the secretion of the C14 defense protease, possibly
during the release or fusion of secretory vesicles to the
EHM (Bozkurt et al., 2011). These findings point toward
a critical role for the control of plant vesicle trafficking
for the establishment of virulence, as shown in animalmicrobe interactions (Baxt et al., 2013). In addition,
oomycete effectors could possibly trigger host membrane reorganization into coalesced membrane rafts,
with a similar mechanism reported for some proteinaceous toxins (García-Sáez et al., 2011). Proteins in the
remorin family were proposed to control PM lateral
organization (Jarsch and Ott, 2011). Their accumulation
in particular membrane domains may facilitate the
action of membrane-targeted effectors or drive the segregation of effectors into specific membrane domains.
Our finding that the AVRblb2 effector colocalizes with
the host susceptibility protein REM1.3 supports the
hypothesis that filamentous plant pathogen effectors
exploit host membrane lateral organization to accommodate infection structures (Bhat et al., 2005; Caillaud
et al., 2012).
Using overexpression of fusion proteins, we observed that REM1.3 showed perihaustorial distribution
in only about half the cases, whereas AVRblb2 always
localized around haustoria. Whether the frequency of
accumulation around haustoria is influenced by the
delivery method remains to be determined. Differential protein accumulation around haustoria may also
be due to dynamic temporal events during haustorial
biogenesis. One possibility is that effectors secreted
from haustoria could mediate the recruitment of
REM1.3 from the EHM or that REM1.3 slowly accumulates in time to reach detectable levels at the EHM.
Therefore, the accumulation of REM1.3 around haustoria may result from selective trafficking toward haustoria or from specific binding to lipids enriched around
haustoria (Perraki et al., 2012).
Differential labeling of REM1.3 and AVRblb2 versus
SYT1 at the EHM shows that, rather than being uniform, the EHM is a patchwork formed by multiple
subdomains. What are the implications of the occurrence of multiple microdomains at the EHM? The
functions of these domains remain unclear. It is possible that these are sites where diverse haustorial activities, such as endocytosis or exocytosis, are regulated
to achieve efficient macromolecule exchange. Future
studies will reveal which endomembrane pathways
contribute to the formation of the EHM microdomains
and uncover the roles that these trafficking pathways
play in plant immunity.
MATERIALS AND METHODS
Plant Lines and Growth Conditions
Leaves from 5-week-old Nicotiana benthamiana and tomato (Solanum lycopersicum ‘Ailsa Craig’) plants grown in a growth chamber at 25°C under 16-hday/8-h-night conditions were used for all experiments. 35S-YFP:REM1.3
transgenic N. benthamiana plants expressing the potato (Solanum tuberosum)
REM1.3 ortholog (StREM1.3) were obtained from Lu et al. (2012), and T2
plants were screened using YFP fluorescence observed with a confocal microscope. Sense and antisense tomato plants misexpressing the tomato
REM1.3 ortholog (SlREM1.2) were obtained from Raffaele et al. (2009). All
tomato plants used were T3 and T4 plants and were screened by protein gelblot analysis using anti-remorin (Raffaele et al., 2009) antibodies. Protein-blot
signal was quantified using the gel analysis function in ImageJ, and only
plants belonging to the bottom and top quartiles for REM1.3 level were considered as antisense and sense plants, respectively (corresponding to remorin
at less than approximately 80% and more than approximately 150% of the
wild-type level, respectively; Supplemental Fig. S3).
Cloning Procedures and Plasmid Constructs
The 35S-YFP:StREM1.3 construct was obtained from Raffaele et al. (2009),
the 35S-RFP:AVRblb2 construct from Bozkurt et al. (2011), the 35S-YFP:
StREM1.3* and 35S-YFP:StREM1.3ΔCA constructs from Perraki et al. (2012),
and the GFP:HaRXL17 construct from Caillaud et al. (2012). The 35S-RFP:
StREM1.3 construct was generated using classical Gateway cloning into the
pH7WGR2 vector (Karimi et al., 2002). The 35S-GFP:SYT1 and 35S-RFP:SYT1
constructs were generated from specific amplification of N. benthamiana
complementary DNAs with the 59-AAAAAGCAGGCTTCATGGGTTTTGTGAGTACTATA-39 and 59-AGAAAGCTGGGTCTCATGATGCAGTTCTCCATTG-39 primers and classical Gateway cloning into the pk7WGF2 and
pH7WGR2 vectors. To design the remorin-silencing construct, we first performed a phylogenetic analysis on Remorin_C domains using remorin sequences identified in the N. benthamiana genome version 0.4.4, the tomato
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Bozkurt et al.
genome International Tomato Annotation Group release 2.3, the potato genome Potato Genome Sequencing Consortium DM 3.4, and the Arabidopsis
(Arabidopsis thaliana) genome (Supplemental Fig. S1A). A 101-amino acid
alignment of a conserved region was constructed using MUSCLE and used as
input in Phylip (Felsenstein, 1989) to build a consensus parsimony tree after a
100-replicate bootstrap analysis. This analysis revealed three orthologs of
REM1.3 in N. benthamiana (Supplemental Fig. S1A). We selected a silencing
construct covering 178 nucleotides at the C terminus of the REM1.3 sequence.
Using homemade perl scripts, we predicted this construct to generate 16 putative 21-nucleotide small interfering RNA species, with three putative targets
in the N. benthamiana genome, corresponding to the three REM1.3 orthologs
(Supplemental Fig. S1B). The VIGS construct was generated by PCR amplification using full-length StREM1.3 as a template with forward primers including a BamHI restriction site and reverse primers including a KpnI
restriction site. PCR products were digested with BamHI and KpnI and ligated into the Agrobacterium tumefaciens binary tobacco rattle virus vector
pTV00 (Ratcliff et al., 2001). Silencing experiments were performed as described (Bos et al., 2010) using pTV00 empty vector as a negative control and
pTV00 carrying the N. benthamiana phytoene desaturase gene fragment as a
silencing control. Remorin silencing was verified by loss of fluorescence in
35S-YFP:REM1.3 stable transgenic plants and anti-remorin western-blot
analysis.
Transient Expression in Planta
A. tumefaciens GV3101 was used to deliver transfer DNA constructs into
3-week-old N. benthamiana plants. Overnight, A. tumefaciens cultures were harvested by centrifugation at 10,000g, resuspended in infiltration medium
(10 mM MgCl2, 5 mM MES, pH 5.3, and 150 mM acetosyringone) prior to syringe
infiltration into either the entire leaf or leaf sections. For confocal microscopy,
constructs were infiltrated to a final optical density at 600 nm (OD600) = 0.4, in
equal amounts in the case of coinfiltrations. For transient protein expression
followed by Phytophthora infestans inoculation, the constructs were infiltrated
to OD600 = 0.3 supplemented with p19 silencing suppressor to OD600 = 0.1, and
P. infestans was inoculated 24 h later. For VIGS silencing, pTV00 and pBINTRA constructs were coinfiltrated at OD600 = 0.3 and OD600 = 0.2, respectively.
Confocal Microscopy
Imaging was performed on a Leica TCS SP5 confocal microscope (Leica
Microsystems) using 203, 403 air, and 633 water-immersion objectives.
REM1.3 localization studies were performed with tagged StREM1.3 constructs. Excitation wavelengths and filters for emission spectra were set as
described (Lu et al., 2012). Colocalization images were taken using sequential
scanning between lines. Image analysis was done with the Leica LAS AF
software, ImageJ (1.43u), and Adobe Photoshop CS4 (11.0). Callose staining
and imaging were performed as described (Bozkurt et al., 2011). The superresolution images were taken on a Zeiss Elyra PS1 structured illumination
microscope using a 633 water objective. The GFP and RFP probes were excited using 488- and 561-nm laser diodes, and their fluorescence emission was
collected at 495 to 550 nm and 570 to 620 nm, respectively. To generate a single
three-dimensional SIM image, 15 raw images were collected (five phases and
three rotations), and the data were processed using Zeiss Zen Black software.
GFP and RFP were collected sequentially, and the SIM images were color
aligned using the channel alignment tool in Zen (calibration beads were taken
at the end of an experiment and used to generate an alignment matrix).
Pathogenicity Assays
Unless stated otherwise, P. infestans infection assays were performed by
inoculation with 10-mL droplets of zoospore solution at 50 zoospores per
microliter on detached N. benthamiana leaves (Chaparro-Garcia et al., 2011).
P. infestans isolate 88069 (van West et al., 1999) and a transformant expressing
a cytosolic tandem DsRed protein (88069td; Whisson et al., 2007) were used.
For transient protein expression followed by P. infestans inoculation, half of the
leaf was infiltrated with A. tumefaciens carrying the 35S-GFP construct as a
control and the other half with a strain carrying the 35S-YFP:StREM1.3 construct. Constructs were expressed by A. tumefaciens-mediated transformation
together with p19 silencing suppressor 24 h prior to P. infestans inoculation.
Lesion sizes were calculated on images taken at 5 dpi, analyzed using area
measurements in ImageJ (1.43u).
1016
Protein Extraction and Immunoblots
Proteins were transiently expressed by A. tumefaciens in N. benthamiana
leaves and harvested 2 d post infiltration. Protein extracts were prepared by
grinding leaf samples in liquid nitrogen and extracting 1 g of tissue in 3 mL of
GTEN protein extraction buffer (150 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10%
(w/v) glycerol, and 10 mM EDTA) and freshly added 10 mM dithiothreitol, 2%
(w/v) polyvinylpolypyrrolidone, 1% (v/v) protease inhibitor cocktail (Sigma),
and 1% (v/v) Nonidet P-40 according to Win et al. (2011). Anti-remorin
(Raffaele et al., 2009) and commercial anti-GFP (Invitrogen) were used as
primary antibodies. Western-blot signal was quantified using gel analysis in
ImageJ (1.43u) and normalized based on the quantification of total proteins
stained by Ponceau Red.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Phylogeny of N. benthamiana remorins and design of a REM1.3 VIGS silencing construct.
Supplemental Figure S2. Characterization of N. benthamiana plants silenced for REM1.3.
Supplemental Figure S3. Details of the molecular and phenotypic characterization of tomato transgenic lines misexpressing the tomato REM1.3
ortholog (SlREM1.2).
Supplemental Data S1. Multiple sequence alignment used for the generation of the parsimony tree in Supplemental Figure S1.
Supplemental Movie S1. Three-dimensional imaging of YFP:REM1.3 with
RFP:AVRblb2 colocalization at the EHM using superresolution microscopy.
Supplemental Movie S2. Three-dimensional imaging of discrete EHM domains marked by RFP:REM1.3 with GFP:SYT1 at the EHM using superresolution microscopy.
Supplemental Movie S3. Three-dimensional imaging of discrete EHM domains marked by RFP:AVRblb2 with GFP:SYT1 at the EHM using
superresolution microscopy.
ACKNOWLEDGMENTS
We thank Joe Win for help with the design of the remorin-silencing
construct, Marie-Cecile Caillaud and Jonathan D.G. Jones for useful comments
and providing the GFP:HaRXL17 construct, Steve Whisson for 88069td, and
Matthew Smoker and Sebastian Schornack for providing N. benthamiana
REM1.3-overexpressing lines. We thank Sebastian Schornack, Joe Win, Liliana
M. Cano, Angela Chaparro-Garcia, Diane G.O. Saunders, Malick Mbengue,
and Silke Robatzek for useful discussions and suggestions.
Received January 15, 2014; accepted May 6, 2014; published May 7, 2014.
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